Duane P. Grandgenett, Ph.D.

ProfessorMolecular Microbiology and Immunology

Education

Ph.D. in Microbiology, University of Iowa, 1970

Research

The Retrovirus Integrase:

Following infection of a cell by a retrovirus and subsequent reverse transcription
of the viral RNA, the linear viral DNA coupled with integrase (IN) and other viral
proteins produce a cytoplasmic macromolecular structure termed the preintegration
complex (PIC). The PIC is transported into the nucleus where the viral DNA ends are
inserted in a concerted fashion by IN into the host chromosomes.

Numerous research laboratories and pharmaceutical companies have made significant
contributions towards understanding the mechanisms involved in retrovirus integration
over the last 38 years. Integration of human immunodeficiency virus type 1 (HIV-1)
DNA into human chromosomes is essential for viral replication. Unchecked replication
of HIV-1 in humans causes AIDS. Integration of the HIV-1 DNA is mediated by the viral
IN. The FDA has approved three effective IN strand transfer inhibitors (STIs) which
are used in combination with other inhibitors directed against the viral reverse transcriptase
and protease to prevent AIDS. As of April 2015, there are five recommended regimens
for antiretroviral therapy for treatment-naïve HIV-1 infected patients: four STI-based
regimens and one ritonavir-boosted protease inhibitor-based regimen (http://aidsinfo.nih.gov/guidelines).

The further development of retrovirus vectors for human gene therapy requires a deep
understanding of the biological and biochemical properties of retrovirus INs.

Current Research Projects

We are currently using purified recombinant HIV-1 and Rous sarcoma virus (RSV) IN
with viral DNA substrates to reconstruct a complex, termed intasome, which mimics
the ability of the PIC to promote concerted integration. The assembly pathways for
the intasome using HIV-1 and RSV IN are being investigated. Clinical HIV-1 strand
transfer inhibitors are also being studied. Crystallography studies of RSV and HIV-1
intasomes as well as IN bound to viral/target DNAs are being pursued. We were recently
successful in determining the atomic structure of the RSV strand transfer complex
that contains viral/target DNA sequences (Nature 530:361-366, 2016).

Research Interests

To investigate the structure-functional relationship between retrovirus IN and its
viral DNA substrate necessary to mediate the biological relevant concerted integration
reaction.

Publications

Major Point: The crystal structure of a three-domain IN from Rous sarcoma virus (RSV)
in complex with viral and target DNAs was determined. The structure shows an octameric
assembly of IN, in which a pair of proximal IN dimers engage viral DNA ends for catalysis
while another pair of distal IN dimers, bridged between the proximal IN dimers, help
capture target DNA. The individual domains of the eight IN molecules play varying
roles to hold the complex together, making extensive networks of protein-DNA and protein-protein
contacts distinct from those previously observed for prototype foamy virus (PFV) intasome,
a tetrameric IN complex. Our work highlights diversity of retrovirus intasome assembly
and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

A movie depicts the RSV intasome structure included in the Nature article. The viral
DNA is black while the target DNA is blue/yellow. The intasome contains eight IN subunits
individually colored. The N-terminal domain (NTD), catalytic core domain (CCD), and
C-terminal domain (CTD) are also depicted. The movie was produced by Dr. Hideki Aihara
(University of Minnesota). v530 n7590.

Major Point: This review examines the multifunctional properties of retrovirus integrase
(IN) besides its key function of integrating the viral DNA into host chromosomes.
IN also has a major role in the maturation of the virus, reverse transcription and
nuclear transport of the preintegration complex. IN binds to cellular cofactors for
uncoating of the viral core and to other cellular proteins that guide the preintegration
complex to prefer regions on the host genome for integration. Understanding these
IN functions has resulted in the production of three clinical IN strand transfer inhibitors
to prevent HIV/AIDS and development of retrovirus vectors for human gene therapy.

A possible role for the asymmetric C-terminal domain dimer of Rous sarcoma virus integrase
in viral DNA binding. Plos One 8:e56892.

Major Point: Crystal structure of the 3-domain Rous sarcoma virus IN was resolved
at 1.86 Å. The binding of Rous sarcoma virus IN to viral DNA may be different than
that observed with the 4-domain prototype foamy virus IN.

Major Point: The HIV-1 IN monomer selectively interacts with the viral DNA ends for
concerted integration and appears to be the precursor of the IN tetramer necessary
for assembly of the synaptic complex (intasome). The monomer may be more suitable
than dimers of IN for producing crystals of IN/DNA complexes.